Regulator of contact-mediated hemolysin

ABSTRACT

The present invention relates to a nucleic acid that encodes a hemolysin protein and to a nucleic acid that encodes a positive regulator of hemolysis. The nucleic acid can be the basis of a vaccine against tuberculosis. The nucleic acid can be inserted into an avirulent vaccine strain such as M. bovis BCG.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a nucleic acid that encodes a hemolysinprotein and to a nucleic acid that encodes a positive regulator ofhemolysis.

2. Background Art

The World Health Organization estimates that more than 2 billion personsworldwide are or have been infected with Mycobacterium tuberculosis, andtuberculosis causes more than 3.5 million deaths annually (1). The humanimmunodeficiency virus (HIV) epidemic has complicated the epidemiologyof tuberculosis (2), and much of the recent increase in tuberculosis indeveloped countries can be traced to the enhanced susceptibility of AIDSpatients to developing this disease (3).

M. tuberculosis is an intracellular pathogen which multiplies withincells of the host's immune system, primarily macrophages (4). Uptake ofthe bacillus by macrophages is thought to be mediated by complementcomponent C3 and complement receptor (5). After entering the cell, M.tuberculosis inhibits phagosome-lysosome fusion (6,7) and theacidification of the phagosome (8). M. tuberculosis then multiplieswithin the unfused vacuole (9). Macrophages heavily ladened with bacilliultimately lyse and release the bacilli.

Studies of other bacterial pathogens have shown that soluble andmembrane-bound cytolysins are important virulence factors. For example,strains of Escherichia coli expressing alpha-hemolysin are 10- to1000-fold more virulent in animal models than strains lackingalpha-hemolysin (10). Similarly, strains of Bordetella pertussis lackingadenylate cyclase/hemolysin display reduced virulence in mouse models(11), and this phenotype can be reversed by trans-complementation with aplasmid expressing the adenylate cyclase/hemolysin (12).

Cytolysins also play important roles in the ability of intracellularbacterial pathogens to enter, replicate within, and exit host cells(13,14,15). The soluble cytolysin of Listera monocytogenes,listeriolysin O, is required for the intracellular growth of thisorganism in macrophages (16,17,18). Expression of listeriolysin O inBacillus subtilis allows this non-pathogen to escape from the phagosomeand multiply within macrophages (19). A membrane-bound cytolysin hasalso been implicated in the escape from phagosomes and intracellulargrowth of Shigella flexneri (20), and more recently the Shigellacytolysin, but not the Listeria cytolysin, has been shown to inducemacrophage cell death through apoptosis (21).

Hemolytic activity in M. tuberculosis, however, has not been studied,due in part to unique difficulties in culturing these cells. Inparticular, these organisms release organic molecules which remain inthe culture medium and cause dumping of the cells, a phenomenon known toscientists in the field. Thus, hemolysis assays would be a practicallydifficult problem with this organism. Furthermore, there has been nocorrelation in the literature between hemolysins and virulence of M.tuberculosis.

The present invention is based in part upon the vital and unexpecteddiscovery that virulent strains of M. tuberculosis possess hemolyticactivity while avirulent strains do not. Such a discovery will helpprovide long-awaited understanding of the mechanisms of infection bythis organism and crucial means of treating and preventing infection anddeath from M. tuberculosis.

The present invention is also based upon the discovery of an E. coligene that regulates hemolysis when placed in any of several differentbacterial organisms. This gene therefore, can be utilized to providegreatly improved vaccine strains against M. tuberculosis as it can aidthese vaccines in causing cell-mediated immunity. Such vaccines aregreatly needed in light of the large numbers of people infected with M.tuberculosis and the devastating effects of infection. Current vaccines,such as the strain M. bovis BCG, have met with only limited success,since, over time after vaccination, protection against tuberculosisdeclines.

SUMMARY OF THE INVENTION

The present invention provides an isolated double-stranded nucleic acidcomprising the sequence set forth in SEQ ID NO:2. This sequence is an E.coli-derived hemolysin regulator. The present invention also providesthe protein encoded by the above nucleic acid, or a biologically activeportion thereof.

The present invention also provides a vaccine comprising a hostcontaining a vector which includes an isolated double-stranded nucleicacid comprising the sequence set forth in SEQ ID NO:2.

Also provided by the instant invention is a method of promoting animmune response in a subject against Mycobacterium tuberculosiscomprising administering to the subject a suitable host, such as M.bovis BCG or M. smegmatis, containing a vector which includes anisolated double-stranded nucleic acid comprising the coding sequencesset forth in SEQ ID NO:2.

The present invention further provides a method of enhancing theimmunogenic effects in a subject of an M. bovis BCG vaccine comprisinginserting a vector which includes an isolated double-stranded nucleicacid comprising the coding sequences set forth in SEQ ID NO:2 into theM. bovis BCG vaccine prior to administering the vaccine to the subject.

The present invention also provides a double-stranded nucleic acidpositively regulated by the protein comprising the polypeptide encodedby SEQ ID NO:2, wherein the nucleic acid encodes a protein havinghemolysis activity.

Also provided is a method of detecting the presence of a virulent strainof Mycobacterium tuberculosis in a sample comprising

(a) identifying the presence of a Mycobacterium tuberculosis nucleicacid sequence in the sample; and

(b) detecting contact-mediated hemolytic activity in the sample, thepresence of Mycobacterium tuberculosis and contact mediated hemolyticactivity indicating the presence of a virulent strain of Mycobacteriumtuberculosis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesincluded therein.

As used herein, "a" or "an" can mean one or more depending on thecontext in which it is used.

The examples presented herein show that the virulent H37Rv strain of M.tuberculosis expresses a cytolytic activity that is significantlyincreased when the bacteria are in close contact with erythrocytes. Arecombinant clone carrying an E. coli genomic locus that causesexpression of a cytolytic activity when transferred into severalorganisms such as M. tuberculosis and M. smegmatis also causesexpression of a cytolytic activity that is significantly increased byclose contact with erythrocytes.

The findings provided herein indicate that M. tuberculosis expresses acontact-dependent cytolysin that is either not expressed or expressedbelow detectable levels in an attenuated strain of M. tuberculosis andan attenuated vaccine strain of M. bovis. These findings also show thatE. coli contains a nucleic acid coding sequence that, when transfectedinto other organisms under the control regulatory sequences compatiblewith that organism, induces a hemolytic phenotype in these organisms.Therefore, this gene is referred to herein as a regulator of hemolysis.

SEQ ID NO:2 sets forth the sequence of the isolated E.coli gene encodinga regulator of hemolysis. This gene encodes a protein found, asdescribed herein, to regulate hemolysis in several bacterial organisms.For example, this sequence, when transfected into M. tuberculosis, M.smegmatis, or E. coli under appropriate regulatory sequences, causes theorganism to demonstrate hemolytic activity. Given this discovery,various therapies can be designed to treat or prevent the hemolysisactivity of M. tuberculosis.

Provided herein is an isolated double-stranded nucleic acid comprisingthe nucleic acid sequence set forth in SEQ ID NO:2. Additionallyprovided is the coding nucleic acid sequence as well as the non-coding,i.e., regulatory region, nucleic acid sequence of this nucleic acid. Asnoted in the sequence listing in SEQ ID NO:2, the non-coding region ofSEQ ID NO:2 includes nucleotides 1-98, and the coding region includesnucleotides 99-1020 or portions thereof. Also provided is the purifiedpolypeptide encoded by the nucleic acid, which amino acid sequence isset forth in SEQ ID NO:3. The invention further provides an isolatednucleic acid encoding this amino acid sequence set forth in SEQ ID NO:3.The nucleic acid can, if desired, include regulatory sequences operablylinked to the coding sequence to control expression of this gene, suchthat the protein can be expressed, for example, by a host which carriesthe gene.

By "isolated" is meant separated from other nucleic acids found in thesource organism. The nucleic acid of SEQ ID NO:2 is genomic sequence andthus includes at least some of the regulatory sequences utilized;however, any other regulatory regions can be operably linked to thecoding sequences for proper expression of the gene in a selectedorganism. "Operably linked" means the sequences are attached such thatthe regulatory regions can direct the expression of the coding region.Therefore, other regulatory regions can be substituted in place of theE. coli regulatory region. For example, the E. coli promoter can bereplaced by an M. tuberculosis promoter, such as the known heatshockhsp60 promoter. Such additional useful regulatory regions are known orcan be found with techniques standard in the art. (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)).

Additionally provided herein is the noncoding region of the nucleic acidof SEQ ID NO:2 operably linked to a reporter gene. Such reporter genesare well known in the art, as are the methods for performing the linkageExamples of such reporter genes include the β-gal gene, LacZ gene andluciferase gene. Such constructs can be useful in screening forcompounds that inhibit or increase expression of this gene. Thus, theregulatory region reporter construct can be useful for screening forcompounds that bind to the regulatory regions of the gene for theregulation of hemolysis. Such screenings could be readily performedaccording to standard methods for screening for expression of a selectedreporter gene, e.g., for detecting the presence of the reporter geneproduct.

Also provided herein is a double-stranded nucleic acid encoding aprotein having hemolysis activity and positively regulated by thepositive regulator of hemolysis described herein. Hemolysis activity canbe assayed utilizing the hemolysis assay described herein. By"positively regulated" is meant that the presence of the regulatorincreases expression of the hemolysin protein's hemolytic activity.

In addition, the invention also provides a nucleic acid encoding aregulator of hemolysin which is homologous to the regulator of hemolysindescribed herein. Such a homologous nucleic acid sequence can be usedfor example to simultaneously detect related strains or as a basis for amultiprotective vaccine. For example, Southern blot analysis indicatesthat a homolog for hemolysin exists at least in M. smegmatis, M.tuberculosis H37Ra, E. coli and M. bovis. However, given the lack ofcytolytic activity expressed by these avirulent bacteria in thehemolysis assay of this invention, this hemolysin gene either is notexpressed, is expressed at undetectable levels, or is mutated such thatits gene product is no longer hemolytic in its activity. Such homologousregulators of hemolysin can be isolated using the procedures set forthherein or by standard procedures utilizing primers and probes derivedfrom the Sequence Listing.

A nucleic acid which selectively hybridizes with the nucleic acidencoding this positive regulator of hemolysis is also contemplated. Asused herein, "selectively hybridizes" means that the nucleic acidspecifically hybridizes to its target (i.e., complementary) nucleic acidbased upon complementarity between the two sequences, rather than randomnon-specific, non-selective hybridization, under suitable stringenthybridization conditions. In other words, the sequences of the nucleicacid utilized for hybridization are unique to the target sequence suchthat the target sequence can be detected apart from other nucleic acidsunder suitable stringent hybridization conditions. Suitably stringentconditions will naturally vary based on the length of the nucleic acids.Thus, a nucleic acid that selectively hybridizes with a nucleic acid ofthe antigen coding sequence will not selectively hybridize understringent conditions with a nucleic acid for a different antigen, andvice versa. The invention provides examples of these nucleic acids, sothat the degree of complementarity required to distinguish selectivelyhybridizing from nonselectively hybridizing nucleic acids understringent conditions can be clearly determined for each nucleic acid.

"Stringent hybridization conditions" refers to the washing conditionsused in a hybridization protocol. In general, the washing conditionsshould be a combination of temperature and salt concentration chosen sothat the denaturation temperature is approximately 5°-20° C. below thecalculated T_(m) of the hybrid under study. The temperature and saltconditions are readily determined empirically in preliminary experimentsin which samples of reference DNA immobilized on filters are hybridizedto the probe or protein coding nucleic acid of interest and then washedunder conditions of different stringencies. For example, hybridizationswith oligonucleotide probes shorter than 18 nucleotides in length can bedone at 5°-10° C. below the estimated T_(m) in 6X SSPE, then washed atthe same temperature in 2X SSPE (see, e.g., Sambrook et al.). The T_(m)of such an oligonucleotide can be estimated by allowing 2° C. for each Aor T nucleotide, and 4° C. for each G or C. An 18 nucleotide probe of50% G+C would, therefore, have an approximate T_(m) of 54° C.

Additionally, the nucleic acids of the invention can have at least 80%homology with the coding nucleotides of SEQ ID NO:2 that are not subjectto the degeneracy of the genetic code, i.e., with the non-"wobble"nucleotides (the wobble nucleotides usually being the third nucleotidein a codon) in the coding sequence. Preferably, the nucleic acids willhave 90%, or more preferably, 95%, or even more preferably, 99% homologywith the coding nucleotides of SEQ ID NO:2 that are not subject to thedegeneracy of the genetic code. The nucleic acids can be at least 18,50, 100, 150, 200, 300, 500, 750 or 1000 nucleotides in length.

Such a nucleic acid can also comprise a primer or probe, for example,that hybridizes to either strand of the nucleic acid and can be used inamplification procedures or in detection of the organism. Such a nucleicacid can also be used, for example, in inhibitory antisense therapy tobind to mRNA molecules transcribed from the coding DNA strand andprevent translation, and to thereby inhibit hemolysis. In addition, theselectively hybridizing nucleic acid can be the length of the entirecoding region and encode a substantially similar protein having thehemolysin activating activity. The sequences of such nucleic acids canbe selected based on the genomic nucleotide sequence and the intendeduse for the particular nucleic acid.

Also provided is a nucleic acid which selectively hybridizes with thedouble-stranded nucleic acid encoding the hemolysin. Thus, this nucleicacid can selectively hybridize, as defined herein, to either strand ofthe nucleic acid encoding hemolysin, and can be used for such functionsas primers, probes, and antisense binding to mRNA transcribed from thenucleic acid to inhibit hemolytic activity or to detect the presence ofM. tuberculosis. This nucleic acid from M. tuberculosis can be readilyascertained by, for example, amplification of homologous sequences usingdegenerative primer(s) PCR (Sadaie, Y. et al., Gene, 98:101-105 (1991);Scaramuzzi, C. D., Current Genetics, 22:421-427 (1992)) and using, forexample, another cytolysin sequence.

Any nucleic acid of the present invention in a vector is also provided.Such a vector can include the nucleic acid as well as additional nucleicacid sequences having numerous functions, such as restrictionendonuclease sites, sequences allowing replication of the vector, markergenes, and other features, known and standard in the art (see e.g.,Sambrook et al.). Typical vectors include numerous plasmids, cosmids andviral constructs. Nucleic acids can be placed into vectors by standardmeans known in the art. Fragments of nucleic acids can also be placedinto vectors, such fragments being generated by known, standardtechniques such as recombinant methods and synthesis methods. Thevectors of the invention can be in a host, particularly a host capableof expressing the protein.

There are numerous E. coli expression vectors known to one of ordinaryskill in the art that are useful for the expression of the antigen.Other microbial hosts suitable for expression use include bacilli, suchas Bacillus subtilus, and other enterobacteriaceae, such as Salmonella,Serratia, and various Pseudomonas species. In these prokaryotic hostsone can also make expression vectors, which will typically containexpression control sequences compatible with the host cell. In addition,any number of a variety of well-known promoters will be present. The DNAsequences can be expressed in hosts after the sequences have beenoperably linked to, i.e., positioned to ensure the functioning of, anexpression control sequence. These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors cancontain selection markers, e.g., tetracycline resistance or hygromycinresistance, to permit detection and/or selection of those cellstransformed with the desired DNA sequences (see, e.g., U.S. Pat. No.4,704,362).

Provided herein is a vector comprising any herein described nucleic acidin a host. Such a "host" can be any organism capable of maintaining thevector within the organism. The host is preferably one that is suitablefor expression of the nucleic acid. Particularly useful hosts areattenuated organisms currently used for vaccine purposes, such as M.bovis BCG, bacteria closely related to virulent M. tuberculosis, such asM. smegmatis, and other bacteria that would maintain copies of thevector such as E. coli. The vector can be readily placed in the host byany of several means, depending upon the type of vector used, such astransfection, transformation, electroporation or microinjection, whichare standard techniques (see Sambrook et al.)

A particularly useful host is one including a vector in which isinserted a nucleic acid encoding the present regulator of hemolysin. SEQID NO:2 provides the genomic sequence of an example nucleic acidencoding this hemolysis regulator which can be inserted into anysuitable vector, as described herein and known in the art, for theselected host, by methods standard in the art. However, any selectednucleic acid sequence encoding the regulator protein can be utilized.For example, due to the degeneracy of the genetic code, the codingsequences can be altered. Furthermore, depending upon the host selected,the regulatory sequences can be modified to select regulatory sequencescapable of allowing expression of the gene in that specific host. Hostscan be selected as described herein. One particularly useful host can beM. bovis BCG, a vaccine strain of mycobacterium. An M. bovis BCG hostcontaining the coding nucleic acid sequences under the control ofsuitable regulatory sequences can be particularly useful for promotingan immune response in a subject. Any selected host and vector can bereadily tested for promoting an immune response by standard immuneresponse testing. This host can be in a pharmaceutically acceptablecarrier.

Each gene may exist as a single contiguous sequence or may, became ofintervening sequences and the like, exist as two or more discontinuoussequences, which are nonetheless transcribed in vivo to ultimatelyeffect the biosynthesis of a protein substantially equivalent to thatdescribed above. Such modifications may be deliberate, resulting from,for example, site directed mutations. Such modifications may be neutral,in which case they result in redundant condons specifying the nativeamino acid sequence or in such modifications which may in fact result ina change in amino add sequence which has either no effect, or only aninsignificant effect on activity of the protein. Retention of theactivity can be readily monitored by the methods taught herein. Suchmodifications may include point mutations, deletions or insertions.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term"nucleic acid encoding for" a protein may thus refer to one or moregenes within a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide whilestill encoding a protein with positive hemolysis regulatory activity orhemolysis activity.

Modifications to the nucleic acids of the invention are alsocontemplated as long as the essential structure and function of thepolypeptide encoded by the nucleic acids is maintained. Likewise,fragments used as primers or probes can have substitutions so long asenough complementary bases exist for specific, selective hybridizationthat distinguishes this gene from other nucleic acids, as describedherein (Kunkel et al. Methods Enzymol. 154:367 (1987)).

Additionally provided herein is a purified protein comprising thepolypeptide whose amino acid sequence is set forth in SEQ ID NO:3, or abiologically active portion thereof. Modifications to the set forthamino acid sequence, such as amino acid substitutions, can be made, asknown in the art, as long as the protein retains its biologicalactivity. This protein has the activity of positively regulatinghemolysis, which activity can be readily detected by the methods taughttherein. By "purified" is meant that the protein is separated from otherproteins in the source organism. By "biologically active portionthereof" is meant a fragment of this protein that still retains itsbiological activity of positively regulating hemolysis, as can readilybe determined. Peptide fragments can be made according to routinemethods known to those of skill in the art, as elaborated upon below.Additionally, modifications to the amino acids typically occurring inthe cell, such as glycosylation and acetylation, can be made.

A protein encoded by a nucleic add that is positively regulated by theinventive regulator of hemolysin, or a hemolyticly active portionthereof, is also provided herein. This protein has hemolytic activity.The nucleic acid encoding this protein can be positively regulated bythe above positive regulator of homolysis, since studies in which thenucleic acid encoding the positive regulator is added to cells thatapparently contain an unexpressed hemolysis structural gene and normallydemonstrate no hemolysis activity, show that the cells subsequentlydemonstrate hemolysis activity.

The complete protein as well as any fragment thereof that retains itsabove-described biological activity is contemplated herein. A"biologically active portion" of any protein herein can also contemplatefragments that retain biological activities such as immunogenicity andimmunoreactivity, as can be determined by standard methods known in theart, as elaborated upon below. A "hemolyticly active portion" of aprotein retains the function of hemolysis, as can be readily tested bythe methods provided herein.

An immunoreactive fragment of any protein provided herein is defined asan amino acid sequence of at least about 5 consecutive amino acidsderived from the protein's amino acid sequence. Such fragments can begenerated, for example, by mechanical or chemical disruption of thecomplete protein or, as another example, they can be recombinantproteins obtained by cloning nucleic acids encoding the polypeptide inan expression system capable of producing the protein or fragmentsthereof. The activity of such fragments can be determined utilizing themethods taught below in the Examples.

The polypeptide fragments of the present invention can also berecombinant peptides obtained by cloning nucleic acids encoding thepolypeptide in an expression system capable of producing the antigenicpolypeptide or fragments thereof.

The present invention also provides an immunogenic amount of a hemolysinprotein of this invention in a pharmaceutically acceptable carrier. Animmunogenic amount can be readily determined by standard methods for thespecific subject to which it is to be administered. Once the amino acidsequence of each protein is deduced from the DNA sequence, it ispossible to synthesize, using standard peptide synthesis techniquesand/or recombinant techniques, peptide fragments that are homologous toimmunoreactive regions of the protein and to modify these fragments byinclusion, deletion or modification of particular amino acid residues inthe derived sequences. Thus, synthesis or purification of an extremelylarge number of peptides derived from the original protein sequence ispossible.

The amino add sequences of the present polypeptides can contain animmunoreactive portion attached to sequences designed to provide forsome additional property, such as solubility. Furthermore, the aminoacid sequences can include sequences in which one or more amino acidshave been substituted with another amino acid to provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its biolongevity, alter enzymaticactivity, or alter interactions with gastric acidity. In any case, thepeptide must posses a bioactive property, such as hemolysin regulation,hemolysis, immunoreactivity, immunogenicity, etc.

The purified polypeptide fragments thus obtained can be tested todetermine their immunogenicity and specificity. Briefly, variousconcentrations of a putative immunogenically specific fragment areprepared and administered to an animal and the immunological response(e.g., the production of antibodies or cell mediated immunity) of ananimal to each concentration is determined. The amounts of antigenadministered depend on the subject, e.g. a human or a guinea pig, thecondition of the subject, the size of the subject, etc. Thereafter ananimal so inoculated with the antigen can be exposed to the bacterium totest the potential vaccine effect of the specific immunogenic fragment.The specificity of a putative immunogenic fragment can be ascertained bytesting sera, other fluids or lymphocytes from the inoculated animal forcross reactivity with other closely related bacteria.

Polynucleotides encoding a variant polypeptide may include sequencesthat facilitate transcription (expression sequences) and translation ofthe coding sequences such that the encoded polypeptide product isproduced. Construction of such polynucleotides is well known in the art.For example, such polynucleotides can include a promoter, atranscription termination site (polyadenylation site in eukaryoticexpression hosts), a ribosome binding site, and, optionally, an enhancerfor use in eukaryotic expression hosts, and, optionally, sequencesnecessary for replication of a vector.

An antibody which specifically binds an antigenic portion of the proteinis also provided for each claimed protein. The antibodies canspecifically bind a unique epitope of the antigen or they can also bindepitopes of other organisms. Thus, the antibodies can be used to detecta particular organism or related organisms. The term "specifically bind"means an antibody specifically binding a protein does not cross reactsubstantially with any antigen other than the one specified, in thiscase, the hemolysin positive regulatory protein or the hemolysinprotein, such that the intended antigen can be detected. Antibodies canbe made by well-known methods, such as described in Harlow and Lane,Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., (1988). Briefly, purified protein, or an antigenicfragment thereof is injected into an animal in an amount and inintervals sufficient to elicit an immune response. Polyclonal antibodiescan be purified directly by passing serum collected from the animalthrough a column to which non-hemolysin regulatory proteins ornon-hemolysin proteins prepared from the same expression system havebeen bound. Monoclonal antibodies can also be produced by obtainingspleen cells from the animal. The cells are then fused with an immortalcell line and screened for antibody secretion. The antibodies can beused to screen DNA clone libraries for cells secreting the antigen.Those positive clones can then be sequenced if desired (see, forexample, Kelly et al., Bio/Technology 10:163-167, (1992) and Bebbingtonet al., Bio/Technology 10:169-175, (1992)).

The antibody can be bound to a substrate or labeled with a detectablemoiety or both bound and labeled. The detectable moieties contemplatedwith the composition of the present invention, include, for example,fluorescent, enzymatic and radioactive markers.

The present invention additionally provides an immunogenic amount of theprotein, i.e., an antigen, in a pharmaceutically acceptable carrier.This composition can include can be the entire antigen, the antigen onan intact avirulent Mycobacterium, E. coli or other strain, or anepitope specific to the antigen. The antigen can also be potentiallycross-reactive with antibodies to other antigens. The composition canthen be used in a method of preventing tuberculosis or othercomplications of M. tuberculosis infection.

Immunogenic amounts of the antigen can be determined using standardprocedures. Briefly, various concentrations of a putative specificimmunoreactive epitope are prepared, administered to an animal and theimmunological response (e.g., the production of antibodies) of an animalto each concentration is determined.

Hosts of this invention can be in a composition with a pharmaceuticallyacceptable carrier, particularly if the host is to be administered to asubject. The pharmaceutically acceptable carrier described in theinstant invention can comprise saline or other suitable carriers (Arnon,R. (Ed.) Synthetic Vaccines I:83-92, CRC Press, Inc., Boca Raton, Fla.,1987). A carrier can be used with this composition of this invention. Anadjuvant can also be a part of the carrier of the host, in which case itcan be selected by standard criteria based on the antigen used, the modeof administration and the subject (Arnon, R. (Ed.), 1987). Methods ofadministration can be by oral or sublingual means, or by injection,depending on the particular host used and the subject to whom it isadministered.

It can be appreciated from the above that the host containing the hereindescribed nucleic acid can be used as a prophylactic or a therapeuticmodality. Thus, the invention provides methods of preventing or treatinginfection and the associated diseases by administering the host havingthe described nucleic acid to a subject.

The present invention further provides a method of promoting an immuneresponse in a subject against Mycobacterium tuberculosis comprisingadministering to the subject a host containing a vector which includesan isolated double-stranded nucleic acid comprising the coding sequenceset forth in SEQ ID NO:2. The administration of the host can stimulatecell-mediated immunity, as well as humoral immunity, to protect againstM. tuberculosis infection. The immune response can be detected bystandard means known in the art. Administration can be performedaccording to standard means known in the art for current M. tuberculosisvaccines, such as M. bovis BCG, including mode of administration anddosages.

Additionally, provided herein is a method of enhancing the immunogenityin a subject of an M. bovis BCG vaccine comprising inserting a vectorwhich includes an isolated double-stranded nucleic acid comprising thecoding sequence of the sequence set forth in SEQ ID NO:2 into the M.bovis BCG vaccine prior to administering the vaccine to the subject.This method can enhance the longevity of effectiveness of standard M.bovis BCG vaccines by stimulating cell-mediated immunity. The enhancedvaccine would then be administered as usual for M. bovis BCG vaccines,at the known doses.

The invention also provides a method of detecting the presence of avirulent strain of M. tuberculosis in a sample comprising identifyingthe presence of a M. tuberculosis nucleic acid sequence in the sampleand detecting contact mediated hemolytic activity in the sample. Thus,avirulent and virulent strains of M. tuberculosis can be distinguished.Such a "sample" can include cultured isolates obtained directly from asubject. A crude lysate of a culture, or a sputum sample for example,can be utilized to detect the presence of M. tuberculosis nucleotide byany several methods known to those of skill in the art (see generally,Sambrook et al.). For example, a nucleic acid specific for M.tuberculosis can be detected utilizing a nucleic acid amplificationtechnique, such as polymerase chain reaction or ligase chain reaction.Alternatively, the nucleic acid is detected utilizing directhybridization or by utilizing a restriction fragment lengthpolymorphism. In addition, PCR primers which hybridize only with nucleicacids specific for M. tuberculosis can be utilized. The presence ofamplification indicates the presence of the M. tuberculosis nucleicacid. In another embodiment a restriction fragment of a DNA sample canbe sequenced directly using, for example, Sanger ddNTp sequencing or7-deaza-2'-deoxyguanosine 5'-triphosphate and Taq polymerase andcompared to known unique sequences to detect M. tuberculosis. Examplesof known M. tuberculosis sequences include the IS6110 insertion sequence(Care et al., Molecular and Cellular Probes, 5:73-80 (1991), the majorpolymorphic tandem repeat (MPTR) sequence (Hermans et al., J.Bact.,174:4157-4165 (1992) and the 65K antigen of M. tuberculosis(Shinnick, T. M., J. Bacteriol., 169:1080-1088 (1987)).

Samples that contain M. tuberculosis DNA can then be analyzed forhemolytic activity by utilizing the hemolysis assay described herein oncell samples. The presence of hemolytic activity in the cell indicates acell that naturally expresses the hemolysin, i.e., a virulent strain.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Polymerase Chain Reaction

Primers were generated to a 1.6 kbp DNA region in the plasmid pTBLA3flanking the hpr gene and the reported sequence of the E. colichromosomal region containing the umuD gene (Walker et al., 1985).Amplification of the E. coli genomic DNA and the plasmid pTBLA3 wasperformed using 10 ul of template DNA (20-100 ng) and 90 ul of areaction mixture consisting of 200 uM (each) deoxy-nucleotidetriphosphates, 1.0 uM (each) primers, 2.5 U Taq polymerase, 10mM-Tris/HCL (pH 8.3), 50 mM-KCL, 1.5 mM-MgCl₂ and 0.01% gelatin assupplied by the Taq polymerase manufacturer (Perkin-Elmer Cetus,Norwalk, Conn.). A three-step cycle of denaturation for 1.45 min at 94°C., annealing for 1.45 min at 55° C., and extension for 3.00 min at 72°C. was performed for 35 cycles on a programmable thermal cycler(Perkin-Elmer Cetus). DNA fragments were sized on a 0.7% TBE agarose gelas described above.

Nucleotide Sequence Analysis

Sequencing was performed using the Prism™ Ready Reaction DyeDeoxy™Terminator cycle sequencing kit and a 373 DNA sequencing systemaccording to the manufacturer (Applied Biosystems, Inc., Foster City,Calif.). M13 forward and reverse primers were used for initialsequencing of the DNA inserts in the plasmids pTBLA3 and pTBLA3/S2, andinternal primers were constructed from these sequence data foroverlapping sequence analysis using a DNA synthesizer (model 381A;Applied Biosystems) in the National Center for Infectious DiseasesBiotechnology Core Facility. Both strands of the E. coli hpr gene andthe DNA region containing the umuD gene upstream to this gene weresequenced independently, and sequence data was edited using the SequenceEditor software package (Applied Biosystems). Sequence analysis anddata-base searches were performed using the GCG Wisconsin sequenceanalysis package (version 7.3, Devereux, 1984) and GenBank (includungrelease 81.0, February 1994).

Hemolytic Activity

M. tuberculosis strains H37Rv (TMC #102) and H37Ra (TMC #201), and M.bovis BCG were originally obtained from the Trudeau Institute, andstored at -70° C. until use. E. coli strain XL1-Blue was purchased fromStratagene Cloning Systems (La Jolla, Calif.) and E. coli strain DH5αwas purchased from Gibco BRL (Grand Island, N.Y.). Prior to eachexperiment, mycobacteria strains were grown as static cultures incomplete Middlebrook 7H9 broth (Difco) at 37° C. for 4 weeks, and E.coli strains were grown as shaken cultures in Luria-Bertani (LB) brothwith or without appropriate antibiotics at 37° C. to stationary phase.

Blood agar plates used to screen the hemolytic phenotype in E. coli andM. smegmatis were prepared by washing citrated whole sheep blood(obtained from the Animal Products Division, CDC) twice with 0.01Mphosphate buffered saline (pH 7.2). Washed sheep blood was then added toTrypticase Soy Agar (TSA) at a concentration of 5% with or without theappropriate antibiotics. E. coli and M. smegmatis transformants wereincubated on this medium at 37° C. for 24 h and 4 days, respectively.

Virulent H37Rv (TMC 102) and avirulent H37Ra (TMC 201) strains ofMycobacterium tuberculosis were screened for contact-dependent lysis ofsheep erythrocytes using a modification of standard erythrocyte lysisassays (20,22). Lysis was monitored by the absorbance of a cell-freesupernatant at 540 nm, which measures the release of hemoglobin fromerythrocytes. Bacilli were harvested by centrifugation (17,500×g) for 10min, washed, and resuspended in 0.1% Tween 80/0.01M phosphate bufferedsaline (PBS) (pH 7.2) at a concentration of ˜1012 bacteria/ml. Sheeperythrocytes were obtained from whole blood by The Animal ProductsDivision, Centers for Disease Control and Prevention, and were washedand resuspended in 0.1% Tween 80/0.01M PBS (pH 7.2) at a concentrationof ˜1010 cells/ml. For co-sedimentation experiments, equal volumes ofthe erythrocyte and bacterial suspensions were mixed, centrifuged at17,500×g for 10 min, and then incubated at 37° C. Mixtures of individualcultures of washed M. tuberculosis and M. bovis strains witherythrocytes were also incubated at 37° C. without sedimentation. After3 h, the samples were centrifuged at 17,500×g for 10 min, thesupernatants were carefully collected, and their absorbance at 540 nm(A₅₄₀) measured. Mean hemoglobin release was calculated by subtractingthe A₅₄₀ of parallel samples containing sheep erythrocytes only thatwere centrifuged and incubated as described above.

When suspensions of the virulent H37Rv bacilli and erythrocytes wereco-sedimented by centrifugation at 17,500×g for 10 min and the pelletsincubated at 37° C. for 3 h, the mean hemoglobin release was A₅₄₀=0.67±0.36. Hemoglobin release was significantly lower (A₅₄₀ =0.19±0.04;p=0.026) when the virulent H37Rv bacilli and erythrocytes were mixed andleft in suspension without centrifugation. In contrast to the virulentH37Rv, the avirulent H37Ra bacilli produced only an A₅₄₀ =0.21±0.16after centrifugation and incubation with sheep erythrocytes, anabsorbance not significantly different from the control lysate(p=0.092). The attenuated vaccine strain M. bovis BCG produced an A₅₄₀=0.06±0.03 after centrifugation and incubation.

Subsequent contact hemolysis experiments using E. coli and E. colirecombinants were performed as described above with two exceptions. PBSwithout Tween 80 was used to wash and resuspend bacteria anderythrocytes, and we used ˜109 bacteria/ml and ˜1010 erythrocytes/ml foreach assay with or without sedimentation.

Screening of Cosmid Library for Contact-Dependent Hemolytic Activity

A cosmid library of the M. tuberculosis H37Rv strain was constructedusing the plasmid pairs pJC98 and pJC100 (24). The plasmids pUC19 andpBlueseript II KS- (Stratagene) were used for subcloning the isolatesconferring contact-dependent cytolysis in E. coli using procedures asdescribed (22).

Overnight cultures of individual transformants of the cosmid library ofM. tuberculosis H37Rv DNA in E. coli K-12 strain XL1-Blue were screenedfor contact-dependent hemolysis of sheep erythrocytes. A single cloneexpressing cytolytic activity, designated pHK101, was isolated from ascreen of 96 transformants. Only stationary cultures of this recombinantwere found to express cytolytic activity. To ensure that thecontact-dependent cytolytic activity of this recombinant, pHK101, wasdue to a gene(s) carried on the cosmid and not an unrelated alterationin the E. coli genome, pHK101 DNA was purified and transformed intofresh competent cells of the nonhemolytic E. coli strains XL1-Blue andDH5α. Both of these new transformants lysed the erythrocytes, whileneither of the naive recipient strains caused any significant lysis.

The contact-dependent cytolytic activity of XL1-Blue (pHK101) cells wascompared with that of XL1-Blue cells containing the plasmids pJC98 orpJC100 which were used to construct the cosmid library. Sedimentation of˜10⁹ XL1-Blue (pHK101) bacteria with erythrocytes produced an A₅₄₀=0.24±0.028, while sedimentation of ˜10⁹ XL1-Blue (pJC98), XL1-Blue(pJC100), or XL1-Blue bacteria with sheep erythrocytes produced an A₅₄₀<0.02. XL1-Blue (pHK101) produced significantly less hemoglobin release(p=0.019) when the bacteria were not centrifuged with sheeperythrocytes. Filtrates of stationary phase cultures of XL1-Blue(pHK101) did not produce significant hemoglobin release (p>0.05).

Genetic Analysis

The cosmid pHK101 contains approximately 32 kb of M. tuberculosis DNA.XbaI fragments of the pHK101 cosmid were subcloned into the XbaI site ofpUC19 and the transformants screened for zones of hemolysis in phosphatebuffered saline-washed sheep erythrocytes embedded in Luria-Bertani-topagar. Only recombinants carrying a 6.5 kb XbaI fragment were surroundedby a ring of lysed erythrocytes. Stationary phase cultures of ˜10⁹ E.coli carrying this plasmid (designated pHK1001) produced a meanhemoglobin release of A₅₄₀ =0.652±0.012, compared with an A₅₄₀ of0.241±0.029 from 10⁹ bacteria carrying the cosmid pHK101. The cytolyticactivity of XL1-Blue transformed with pHK1001 was also restricted to thestationary phase of growth, and culture filtrates from stationary phasecultures did not induce lysis of sheep erythrocytes. The cytolyticactivity was further localized to a 3.2 kb NotI fragment from theplasmid pHK1001 by cloning this fragment into pBluescript II KS⁻. Thisplasmid was designated pTBLA3. This fragment hybridized to identicallysized NotI fragments in genomic DNAs from M. tuberculosis H37Rv, M.tuberculosis H37Ra, and M. bovis BCG. These regions may representhomologs of the regulator gene in these organisms.

Genetic Analysis of Hemolysin Induction by hpr in E. coli and M.smegmatis

Initial analysis of the gene responsible for this hemolytic phenotype inE. coli was performed by subcloning the 1,978 bp Not I/Sal I fragmentfrom the plasmid pTBLA3/S2 (King et al., 1994), and screening subclonesfor hemolysis on blood agar plates. The plasmid pTBLA3/S2 was digestedwith a Not I/Sal I double digest and the 1,978 bp fragment was purifiedfrom a 1.0% Tris-borate-EDTA (TBE) agarose gel using Geneclean II (Bio101, LaJolla, Calif.) according to the manufacturers' directions. ThisDNA fragment was then cleaved into two fragments using a Bgl II digestidentified after sequence analysis of this DNA fragment. This generatedan 852 bp fragment with Bgl II/Not I sites and a 1,126 bp fragmentcontaining Bgl II sites. These two fragments were purified from a 1% TBEagarose gel as described above, ligated separately into the BamH I orBamH I/Not I sites of pBluescript II^(ks-) and transformed into E. colistrain XL1-Blue. All transformants were screened for zones of hemolysison blood agar plates containing 50 μg/ml ampicillin and incubated at 37°C. for 24 h.

Partial deletion of the umuD gene contained in the 1,978 bp fragment wasperformed to determine the importance of its role in the induction ofhemolysin in E. coli and M. smegmatis. A 404 bp fragment containing a151 bp deletion of the umuD gene was cloned out of the plasmid pTBLA3/S2by digestion with the enzyme HinC II, and purification and relegation ofthe remaining gene sequence to the purified parent vector was performedas described above. The 404 bp fragment containing the 151 bp partiallydeleted umuD gene was also purified and ligated back to the parentplasmid. Both subclones were screened for zones of hemolysis on bloodagar plates as described above. The ability of the reported umuD genesequence to complement this phenotype in E. coli was then tested bycloning the plasmid pSE117 containing the umu operon (Walker, 1984) intoE. coli XL1-Blue. Transformants containing this plasmid were then testedwith the contact-hemolysin assay and for hemolysis on blood agar platesas described above.

The 1,978 bp fragment from the plasmid pTBLA3/S2 was also analyzed forthe presence of an independent promoter for the transcription of hpr inE. coli. This was done by subcloning the 1,978 bp EcoR I/Sal I fragmentfrom the plasmid pTBLA3/S2 into the EcoR I/Sal I sites of the plasmidspUC18 and pUC19. These clones were then screened for the hemolyticphenotype on blood agar plates containing 50 ug/ml ampicillin, andinsert orientations were confirmed by sizing the DNA fragments on a 0.7%TBE agarose gel after digestion of the plasmids with the enzymes NdeI/Cla I. The enzyme Nde I cuts the plasmids pUC19 and pUC18 once and theCla I enzyme only cuts once within the 1,978 bp insert.

Expression in Mycobacterium smegmatis

Evaluation of the induction of a hemolytic phenotype in M. smegmatis wasperformed by cloning the DNA fragments that induced this phenotype in E.coli into the electroporatable M. smegmatis strain LR222. The 3.2 kbpBamHI fragment from the plasmid pTBLA3 (King et al., 1993) containinghpr was cloned into E. coli strain XL1-Blue using the BamHI sites of theE. coli/mycobacterial shuttle vector pMV261 (Stover et al., 1991) andthis plasmid was designated pMV261/S3. The plasmid pMV261 was alsocloned into E. coli strain XL1-Blue without insert for a plasmidcontrol. Purified plasmids from E. coli were transformed into M.smegmatis strain LR222 and the transformants were plated on blood agarplates containing 50 μg/ml kanamycin, incubated at 37° C. for four days,and scored for zones of hemolysis.

Plasmids pMV261/S3 and pMV261 were purified from the hemolytic andnonhemolytic M. smegmatis clones, respectively. The DNA insert in theplasmid pMV261/S3 was confirmed by cloning the plasmid back into E. colistrain XL1-Blue, purifying the plasmid from hemolytic E. coIitransformants, and sizing the insert on a 1.0% TBE agarose gel afterdigestion with the enzyme BamHI. Hemolytic and nonhemolytic M. smegmatisand E. coli clones on blood agar plates were also tested using thecontact-dependent hemolysis assay.

Then it was determined if the putative promoter to hpr was functional inthe induction of the hemolytic phenotype in M. smegmatis by cloning the1,978 bp fragment containing hpr into M. smegmatis in oppositeorientations to the mycobacteria promoter on the plasmid pMV261. Theenzyme Sal I was used to isolate the 1,978 bp fragment with Sal I endsfrom the plasmid pTBLA3 and cloned into E. coli XL1-Blue using the Sal Isite of the vector pMV261. The plasmids of several hemolytic E. colitransformants were screened for the two different Sal I orientations inthe vector pMV261 by digestion with enzyme Cla I and sizing thefragments on a 1.0% TBE agarose gel as described above. Bothorientations were presumed to induce hemolysin in E. coli due to theindependently functioning promoter region located upstream from the hprgene on this fragment. The pMV261 plasmids containing both orientationsof the Sal I fragment (designated pMV261/S2 and pMV261/S2°) werepurified and then electroporated into M. smegmatis strain LR222. M.smegmatis transformants were screened for hemolysis on blood agar platescontaining kanamycin, and plasmids were confirmed by purification andcloning back into E. coIi as described above.

Minicell Analysis

The plasmids pMV261, pMV261/S3 and pMV261/S2 were cloned separately intothe E. coli minicell strain 678-54 (Alder et al., 1967) and hemolyticand nonhemolytic clones confirmed using blood agar plates and thecontact-dependent hemolysis assay. Minicells were isolated from these E.coli clones using as previously described (Quinn and Tompkins, 1989)after growing these clones to early stationary phase in LB mediumcontaining the 50 ug/ml kanamycin. ³⁵ S!-methionine labeled proteinswere analyzed on a 10% SDS-PAGE along with protein standards. The gelswere stained with Coomassie blue, fixed with En³ hance solutionaccording to the manufacturer (Dupont, Boston, Mass.), and dried downonto filter paper. Gels were exposed to Kodak AR X-ray film for 24 hoursat -70° C.

The E. coli subclone containing a 3.2 kbp Not I fragment from the cosmidpHK101 was found to produce clear zones of hemolysis on blood agarplates containing washed sheep erythrocytes, but no zones of hemolysiswere produced by this clone on blood agar plates prepared with wholeblood. This subclone also produced a contact-dependent hemolysis ofsheep erythrocytes similar to that of the E. coli clone containingpHK101. When this 3.2 kbp fragment was cloned into E. coli using thevector pMV261 (plasmid designated pMV261/S3) and then purified andcloned from a hemolytic E. coli transformant into the M. smegmatisstrain LR222, this DNA fragment was also found to induce zones ofhemolysis in mycobacterial transformants after growth on blood agarplates incubated for 4 days. E. coli and M. smegmatis transformantscontaining the vector pMV261 were nonhemolytic after 4 days culture onblood agar plates and M. smegmatis transformants remained negative after14 days incubation. Although both E. coli and M. smegmatis transformantscontaining the vector pMV261/S3 were hemolytic on blood agar plates,only the E. coli transformants produced contact-dependent hemolysis ofwashed sheep erythrocytes.

Size analysis of the DNA insert in the plasmid pMV261/S3 was performedon the plasmid after replication in E. coli and M. smegmatis. Thesestudies demonstrated that a deletion within the 3.2 kbp BamH I fragmentoccurred when this plasmid was cloned into M. smegmatis. The supercoiledplasmid isolated directly from M. smegmatis was approximately 6.0 kbpand the BamH I fragment isolated from this plasmid after transformationand replication in E. coli was 1.5 kbp.

Partial sequence analysis and data base search of the 3.2 kbp fragmentin the plasmid pTBLA3 demonstrated that this fragment containedhomologous regions of the umuD and umuC genes which matched 100% to theDNA sequence on the E. coli chromosomal region containing the umu operonreported by Kitagawa et al., 1985 and Perry et al., 1985. Subcloning ofthis DNA insert was performed to determine which gene located on thisinsert encoded the hemolytic phenotype in E. coli and M. smegmatis. A1,978 bp Sal I/Not I fragment subcloned from the plasmid pTBLA3 into thevector pMV261 was found to induce hemolysis of E. coli and M. smegmatistransformants. Partial sequence analysis of this 1,978 bp insert in theplasmid pTBLA3/S2 demonstrated that in addition to the partial sequenceof umu operon, this fragment also contained an unreported 852 bp DNAsequence upstream from the homologous Bgl II site of the reported DNAsequence for the chromosomal region containing umuD.

Subclone analysis of this 1,978 bp insert indicated that the umuD genewas not responsible for induction of the hemolytic phenotype in E. colior M. smegmatis. A Bgl II fragment subcloned from the 1,978 bp fragmentcontaining the intact umuD gene and its promoter cloned into the vectorpBluescript II^(ks-) was unable to confer a hemolytic phenotype to E.coli transformants. E. coli transformants carrying the Bgl II/Not Ifragment from this insert were also nonhemolytic. Partial deletion ofthe umuD gene from the plasmid pTBLA3/S2 had no effect on the hemolyticphenotypes of E. coli transformants carrying this plasmid. E. coliclones containing the deleted portion of umuD and partial sequence ofumuC were nonhemolytic. In addition, the same E. coli strainstransformed with the plasmid pSE117 which contains the clonedchromosomal regions of the E. coli umu operon were nonhemolytic on bloodagar plates or by the contact-dependent hemolysin assay.

DNA amplification of the E. coli strain XL1-Blue chromosome usingprimers constructed to a flanking region of the umuD gene and theunreported DNA sequence located upstream to umuD on the 1,978 bpfragment resulted in the amplification of an identically sized fragmentof 1.6 kbp from the genomic DNA of E. coli and the plasmid pTBLA3 whensized on a 0.7% TBE agarose gel.

Opposite orientations of the 1,978 bp fragment in relation to the LacZpromoter in the vectors pUC18 and pUC19 had no effect on the hemolyticphenotypes of E. coli transformants containing these plasmids indicatingthe presence of an independent promoter that was functional in E. coli.A Sal I fragment containing this insert was ligated into oppositeorientations with respect to the hsp60 promoter in the Sal I site of thevector pMV261 and these two plasmids, pMV261/S2 and pMV26152°, werecloned into E. coli strain XL1-Blue. This placed the transcription ofthe gene on this DNA insert in opposite orientations with respect to thehsp60 promoter in pMV261 and was done to determine if the independentpromoter was functional in M. smegmatis, E. coli transformantscontaining pMV261/S2 and pMV261/S2° were equally hemolytic on blood agarplates. In contrast, only M. smegmatis transformants containing theplasmid pMV261/S2° were hemolytic. These results demonstrate that theindependent promoter for the gene that induced hemolysin in E. coli wasnot functional in M. smegmatis. Orientation of this gene under controlof the hsp60 promoter in pMV261 did induce the hemolytic phenotype in M.smegmatis demonstrating that transcription of the hpr gene on the 1,978bp fragment that induced hemolysin was opposite to the homologous DNAsequence containing the umuD gene. Hemolytic clones of M. smegmatiscontaining pMV261/S2° were also found to form large aggregates of cellsduring cultivation in 7H9 and TSA broth compared to nonhemolytic clonescontaining pMV261.

Complete sequence analysis of the 1,978 bp fragment confirmed thepresence of a second open reading frame believed to encode hpr on theopposite strand and in the opposite orientation to the homologous DNAsequence containing umuD. This hpr sequence is set forth in SEQ ID NO:2(1021 bp). The orientation of the sense strand containing this openreading frame was in agreement with the expression of a hemolyticphenotype induced by this gene in M. smegmatis using the vector pMV261as shown above. No other continuous open reading frames of greater than50 amino acids were found on this DNA fragment. The DNA sequencecontains an overall G+C composition of 40.8% which is atypical for E.coli DNA sequences (Marmur and Doty, 1962). There is a 5/6 match of DNAsequence located 17 bp upstream to the putative start codon of hpr withthe -10 consensus sequence for E. coli promoters (TATAAT), and a 4/6match of sequence to the -35 consensus sequence (TTGACA) located 46 bpupstream to this codon. There is also a possible ribosome binding sitethat has a 4/8 match to the E. coli consensus sequence (AGGAAAGG) 9 bpupstream from the putative ATG codon.

If the putative ATG codon at position 99 of the DNA sequence for thisopen reading frame is assumed to be the initiator codon and a stop codonis located in frame to this open reading frame after the Not I site onthe cloning vector, then this open reading frame encodes a protein of309 amino acids ending with an alanine. The predicted molecular weightfor this protein based on the DNA sequence is approximately 34,000daltons. It was unusual that this open reading frame did not contain astop codon, but the predicted molecular weight of the protein encoded byhpr using the vector stop codon sequence matched the actual proteinexpressed in E. coli minicells containing this gene. Hemolytic clones ofE. coli minicells containing pMV261/S2° expressed an approximately34,000 dalton protein whereas nohemolytic E coli minicells containingpMV261 did not produce this protein. This suggests that the stop codonlocated on the vector sequence and in frame to the hpr open readingframe was functional for termination of the hpr gene and that this genewas required for induction of the hemolytic phenotype in these E coliclones.

The DNA sequence containing the putative open reading frame for hpr wassearched for homology at the DNA and amino acid level. The DNA sequencecontained a 62.0% homology to the Chlamydial hctA gene over a 129 bpregion. This gene has been described to encode a lysine-richhistone-like DNA binding protein involved in nucleoid condensation(Hackstadt et al., 1991). The hpr gene also had a 55.9% homology to theActinobacillus pleuropneumoniae apxI operon with a 102 bp regioncontaining the open reading frame for the apxIA structural gene forhemolysin. In addition, there was a 62.0% homology over a 100 bp regionand a 53.3% homology over a 225 bp region for the Lactococcus lactisLacR gene and the M. gallisepticum ATP operon respectively.

The ability to lyse membranes in other bacteria suggests that cytolysinsplay roles in processes such as invasion or entry into eukaryotic cells,intracellular multiplication, cell-to-cell spread, or escape frommembrane-bound vacuoles or cells (Mims, C. A. et al., The Pathogenesisof Infectious Disease, Academic Press, Inc., San Diego, Calif. (1990)).The genomic locus for contact-dependent cytolysis in the virulent M.tuberculosis strain H37Rv is homologous to genomic regions in theattenuated strains M. tuberculosis H37Ra and M. bovis BCG, and thus,these strains may be attenuated due to their lack of or loweredexpression of this cytolytic activity. The hybridization andamplification data also indicates that there is an M. tuberculosishomolog to the E. coli umuCD operon.

Another possible role for this cytolytic activity may be in the releaseof mycobacteria from heavily laden macrophages. Such a role would becompatible with the observation that only E. coli recombinants fromstationary-phase cultures express this cytolytic activity. The possibleroles of cytolysins in pathogenesis are not limited simply to the lysisof membranes. Even in the absence of lysis, bacterial cytolysins canexert profound pleiotropic effects on eukaryotic cells (for a review seeWelch, R. A. et al., Infect. Immun., 43:156-160 (1991)). For example,energy metabolism may be disrupted (Bhakdi, S. et al., J. Clin. Invest.,85:1746-1753 (1990)) or the ability to respond to exogenous signals maybe impaired (Welch, R. A. et al., The Molecular Biology of MicrobialPathogenicity, Academic Press, Inc., New York (1986).

Therefore, the gene for the regulator of hemolysin, e.g., as listed inSEQ ID NO:2, can be the basis of a vaccine against tuberculosis. Thegene can be inserted into an avirulent vaccine strain such as M. bovisBCG, by means known and standard in the art. This modified virus canthen be administered as a vaccine, following methods and dosagescurrently used to administer M. bovis BCG as a vaccine. The inventivevaccine can infect macrophages, as do the prior M. bovis BCG vaccines;however, the inventive vaccine, with its hemolysin regulator gene, canhave the added ability to escape phagosomes into the cytoplasm, forpresentation of antigens and resultant cell-mediated immunity.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1023 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: E. coli                                                         (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: pTBLA3                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 679..682                                                        (D) OTHER INFORMATION: /note= "/product=Translation start                     codon for umuD coding sequences"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCGACATCACCCGATTGATCATGTTGCGTGAAGCTCATAATTGCTGCTAAAGCAAACCA60                CGCCACAGCGACGAAACAGATCTTTTTGTTTGAACCAGGGATCGCCCATTTTAACGCCAA120               GCGCCTTTGCCTCAGTGTTTCGGGCGATAACGCAACCGTCATTATTCGATAGCACAACCA180               CCGGTTTACCCCATAAATCAGGGCGAAACACCGTCTCACAGCTGGCATAAAACGCGTTTA240               CATCACAGAGGGCAAACATCAGCGCATCGCCTTAAGACGTGGTTCACCACACCAAAAGAC300               ATCCAGCGTATCTTCACTACTGATGGGTAATGGGCGGAGTACGCGCTGTTCATGGGAATA360               AGCTGTACCGTCGGGCGTAGTTGCAATTTTTTCACCGTAAACTCGCCGTCAACAGCAGCG420               ATGACAATATCACCATGGCTGGCGGTAATAGCGCTATCGACAATCAGTAAATCACCGTCA480               CTAATTCCACCATCAATCATAGAATCACCACTTGCTTTGACGAAGTAAGTCGCGCTGGGA540               TGCTGGATCAACAGTTGATTCAGATCGATGCGCTGTTCAACGTAATCTGCTGCCGGTGAA600               GGAAAGACCACACTGAACAAGATCGCTTAAATAGCGGAAAAGTCACAATTTCGCGGAGAT660               CCGCAGGCTTGATAAACAACATAATAATCTGCCTGAAGTTATACTGTTTTTATATACAGT720               AGTCTGTTCTTGCCAGCAGATCAATACTGATTCAGGCTATCAATATTTGTCGCTGCATAG780               GCTGCTGATTTTTCGTTCTCTTATCTTGTGCTCACGTGGCCTTCTGGCGACGACGCTCAT840               CCAGCAGAAATGAAAAATATCACCCGGCTAAAAAATAGAATAGAAGCATCGCCATAATGA900               CATTAAACATTGTTTGGATATTTATCATATTTAATAGAAATAAAGACATTGACGCATCCC960               CGCCCGGCTTAACTATGAATTAGATGAAGTAAAATTTATTAATAGTTGTAAAACAGGAGT1020              TTC1023                                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1020 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: E. coli                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 99..101                                                         (D) OTHER INFORMATION: /note= "Translation start codon"                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 76..81                                                          (D) OTHER INFORMATION: /note= "5/6 match with -10                             consensus sequence for E. coli promoters (TATAAT)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 47..52                                                          (D) OTHER INFORMATION: /note= "4/6 match with -35                             consensus sequence (TTGACA)"                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 87..94                                                          (D) OTHER INFORMATION: /note= "4/8 match with the E. coli                     consensus sequence for ribosome binding sites (AGGAAAGG)"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCCGCCCGGCTAACTATGAATTAGATGAAGTAAAATTTATTAATAGTTGTAAAACAGGAG60                TTTCATTACAATTTATATATTTAAAGAGGCGAATGATTATGACTGAAATCGTTGCAGATA120               AAACGGTAGAAGTAGTTAAAAACGCAATCGAAACCGCAGATGGAGCATTAGATCTTTATA180               ATAAATATCTCGATCAGGTCATCCCCTGGCAGACCTTTGATGAAACCATAAAAGAGTTAA240               GTCGCTTTAAACAGGAGTATTCACAGGCAGCCTCCGTTTTAGTCGGCGATATTAAAACCT300               TACTTATGGATAGCCAGGATAAGTATTTTGAAGCAACCCAAACAGTGTATGAATGGTGTG360               GTGTTGCGACGCAATTGCTCGCAGCGTATATTTTGCTATTTGATGAGTACAATGAGAAGA420               AAGCATCCGCCCAGAAAGACATTCTCATTAAGGTACTGGATGACGGCATCACGAAGCTGA480               ATGAAGCGCAAAAATCCCTGCTGGTAAGCTCACAAAGTTTCAACAACGCTTCCGGGAAAC540               TGCTGGCGTTAGATAGCCAGTTAACCAATGATTTTTCAGAAAAAAGCAGCTATTTCCAGT600               CACAGGTAGATAAAATCAGGAAGGAAGCATATGCCGGTGCCGCAGCCGGTGTCGTCGCCG660               GTCCATTTGGATTAATCATTTCCTATTCTATTGCTGCGGGCGTAGTTGAAGGAAAACTGA720               TTCCAGAATTGAAGAACAAGTTAAAATCTGTGCAGAATTTCTTTACCACCCTGTCTAACA780               CGGTTAAACAAGCGAATAAAGATATCGATGCCGCCAAATTGAAATTAACCACCGAAATAG840               CCGCCATCGGTGAGATAAAAACGGAAACTGAAACAACCAGATTCTACGTTGATTATGATG900               ATTTAATGCTTTCTTTGCTAAAAGAAGCGGCCAAAAAAATGATTAACACCTGTAATGAGT960               ATCAGAAAAGACACGGTAAAAAGACACTCTTTGAGGTACCGAGCTCGAATTCCCCGGATG1020              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetThrGluIleValAlaAspLysThrValGluValValLysAsnAla                              151015                                                                        IleGluThrAlaAspGlyAlaLeuAspLeuTyrAsnLysTyrLeuAsp                              202530                                                                        GlnValIleProTrpGlnThrPheAspGluThrIleLysGluLeuSer                              354045                                                                        ArgPheLysGlnGluTyrSerGlnAlaAlaSerValLeuValGlyAsp                              505560                                                                        IleLysThrLeuLeuMetAspSerGlnAspLysTyrPheGluAlaThr                              65707580                                                                      GlnThrValTyrGluTrpCysGlyValAlaThrGlnLeuLeuAlaAla                              859095                                                                        TyrIleLeuLeuPheAspGluTyrAsnGluLysLysAlaSerAlaGln                              100105110                                                                     LysAspIleLeuIleLysValLeuAspAspGlyIleThrLysLeuAsn                              115120125                                                                     GluAlaGlnLysSerLeuLeuValSerSerGlnSerPheAsnAsnAla                              130135140                                                                     SerGlyLysLeuLeuAlaLeuAspSerGlnLeuThrAsnAspPheSer                              145150155160                                                                  GluLysSerSerTyrPheGlnSerGlnValAspLysIleArgLysGlu                              165170175                                                                     AlaTyrAlaGlyAlaAlaAlaGlyValValAlaGlyProPheGlyLeu                              180185190                                                                     IleIleSerTyrSerIleAlaAlaGlyValValGluGlyLysLeuIle                              195200205                                                                     ProGluLeuLysAsnLysLeuLysSerValGlnAsnPhePheThrThr                              210215220                                                                     LeuSerAsnThrValLysGlnAlaAsnLysAspIleAspAlaAlaLys                              225230235240                                                                  LeuLysLeuThrThrGluIleAlaAlaIleGlyGluIleLysThrGlu                              245250255                                                                     ThrGluThrThrArgPheTyrValAspTyrAspAspLeuMetLeuSer                              260265270                                                                     LeuLeuLysGluAlaAlaLysLysMetIleAsnThrCysAsnGluTyr                              275280285                                                                     GlnLysArgHisGlyLysLysThrLeuPheGluValProSerSerAsn                              290295300                                                                     SerProAspAlaAla                                                               305                                                                           __________________________________________________________________________

What is claimed is:
 1. The isolated double-stranded nucleic acid setforth in SEQ ID NO:2.
 2. The nucleic acid of claim 1 in a vector.
 3. Anisolated nucleic acid comprising nucleotides 99-1020 of the nucleic acidof claim
 1. 4. An isolated nucleic acid comprising SEQ ID NO:2.
 5. Thenucleic acid of claim 4 operably linked to a reporter gene.
 6. Thenucleic acid of claim 5 in a vector.
 7. The vector of claim 6 in a hostsuitable for expression of the nucleic acid.
 8. An isolated nucleic acidencoding a purified protein comprising the polypeptide set forth in SEQID NO:3.
 9. A vector comprising the nucleic acid of claim
 8. 10. Thevector of claim 9 in a host suitable for expression of the nucleic acid.11. The vector of claim 10, wherein the host is M. bovis BCG.
 12. Thevector of claim 10, wherein the host is Mycobacterium smegmatis.
 13. Amethod of promoting an immune response in a subject againstMycobacterium tuberculosis comprising administering to the subject thehost of claim
 11. 14. A method of promoting an immune response in asubject against Mycobacterium tuberculosis comprising administering tothe subject the host of claim
 12. 15. A method of enhancing theimmunogenicity of an M. bovis BCG vaccine in a subject: comprisinginserting the vector of claim 9 into the M. bovis BCG vaccine prior toadministering the vaccine to the subject.